© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 842-854 doi:10.1242/dev.090449
RESEARCH ARTICLE
Oocyte polarity requires a Bucky ball-dependent feedback
amplification loop
ABSTRACT
In vertebrates, the first asymmetries are established along the
animal-vegetal axis during oogenesis, but the underlying molecular
mechanisms are poorly understood. Bucky ball (Buc) was identified
in zebrafish as a novel vertebrate-specific regulator of oocyte polarity,
acting through unknown molecular interactions. Here we show that
endogenous Buc protein localizes to the Balbiani body, a conserved,
asymmetric structure in oocytes that requires Buc for its formation.
Asymmetric distribution of Buc in oocytes precedes Balbiani body
formation, defining Buc as the earliest marker of oocyte polarity in
zebrafish. Through a transgenic strategy, we determined that excess
Buc disrupts polarity and results in supernumerary Balbiani bodies in
a 3′UTR-dependent manner, and we identified roles for the buc
introns in regulating Buc activity. Analyses of mosaic ovaries indicate
that oocyte pattern determines the number of animal pole-specific
micropylar cells that are associated with an egg via a close-range
signal or direct cell contact. We demonstrate interactions between
Buc protein and buc mRNA with two conserved RNA-binding proteins
(RNAbps) that are localized to the Balbiani body: RNA binding protein
with multiple splice isoforms 2 (Rbpms2) and Deleted in
azoospermia-like (Dazl). Buc protein and buc mRNA interact with
Rbpms2; buc and dazl mRNAs interact with Dazl protein.
Cumulatively, these studies indicate that oocyte polarization depends
on tight regulation of buc: Buc establishes oocyte polarity through
interactions with RNAbps, initiating a feedback amplification
mechanism in which Buc protein recruits RNAbps that in turn recruit
buc and other RNAs to the Balbiani body.
KEY WORDS: Bucky ball, Oocyte polarity, Balbiani body, Rbpms2
INTRODUCTION
The vertebrate animal-vegetal axis is established during oogenesis,
whereas the anteroposterior and dorsoventral embryonic axes arise
after fertilization. Oocyte polarity is a prerequisite for determining
the prospective embryonic axes and germ cell determination in some
non-mammalian vertebrates. Oocyte polarity can be first
distinguished histologically by the asymmetric distribution of
organelles, proteins and mRNAs within the Balbiani body (Bb) (de
Smedt et al., 2000; Kloc et al., 2004; Marlow, 2010; Pepling et al.,
1
Department of Developmental and Molecular Biology, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. 2Department of
Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
NY 10461, USA. 3Dominick P. Purpura Department of Neuroscience, Albert
Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
*Present address: Deutsches Zentrum für Neurodegenerative Erkrankungen
(DZNE) – München Adolf Butenandt-Institute of Biochemistry Ludwig-Maximilians
University Munich, Schillerstrasse 44, 80336 Munich, Germany.
‡
Author for correspondence ([email protected])
Received 6 October 2012; Accepted 2 December 2013
842
2007). The Bb is an evolutionarily conserved asymmetric structure
that is present in early oocytes of all animals examined, including
humans. The Bb is a transient structure assembled in primary
oocytes and disassembled thereafter. In zebrafish and Xenopus the
Bb is the first indicator of the vegetal pole. The relationship between
the Bb and the animal-vegetal axis of mammalian oocytes is not
known. Despite its conserved structure and status as the first
asymmetric structure in oocytes, only one gene, bucky ball (buc), is
known to be required for Bb assembly in vertebrates (Bontems et
al., 2009; Dosch et al., 2004; Kloc et al., 2004; Marlow, 2010;
Marlow and Mullins, 2008). In zebrafish oocytes, Bb assembly in
primary oocytes [stage Ia (zygotene), Ib (diplotene of meiosis I)]
requires Buc (Bontems et al., 2009; Marlow and Mullins, 2008) and
its disassembly in stage II oocytes requires Magellan (Mgn; Macf1
– ZFIN), a microtubule-actin crosslinking factor (Gupta et al., 2010).
Proper regulation of Bb development is essential to establish the
animal-vegetal axis and deliver RNAs and proteins to the vegetal
pole. Three pathways that localize RNAs are known in vertebrate
oocytes: transit through the Bb pathway, utilization of the ‘late
vegetal pathway’, and an animal pole transport pathway (Abrams
and Mullins, 2009; Gagnon and Mowry, 2011; Kloc et al., 2001;
Kloc and Etkin, 1995; Kloc and Etkin, 2005; Kloc et al., 1998;
Marlow, 2010; Zhou and King, 2004). Mutations that ablate the Bb
(buc) (Bontems et al., 2009; Marlow and Mullins, 2008) or block its
disassembly (mgn) (Gupta et al., 2010) disrupt localization of
mRNAs along the animal-vegetal axis. The resulting eggs lack
animal-vegetal polarity (Bontems et al., 2009; Marlow and Mullins,
2008). In zebrafish, asymmetry is also evident in the fates of the
somatic follicle cells. At the animal pole of WT oocytes a single
somatic cell forms the micropyle, a channel on the eggshell required
for fertilization. buc mutant eggshells have excess micropyles,
which leads to polyspermy (Marlow and Mullins, 2008).
Nonsense mutations disrupting buc uncovered a role for Buc
protein in promoting Bb assembly, but the regulation and function
of buc during Bb assembly are not understood. Although Buc
protein lacks identifiable functional domains, the dynamic
localization of buc gene products in the Bb and later at the animal
pole cortex (Bontems et al., 2009) suggests that localizing buc
mRNA might be an important aspect of Buc regulation. Transcripts
of the Xenopus homolog of the buc gene, Xvelo, localize to the
oocyte vegetal pole and the relevant cis-acting elements in the Xvelo
3′UTR are known (Claussen and Pieler, 2004; Mowry and Melton,
1992). buc mRNA is not properly localized in buc mutants
(Bontems et al., 2009; Marlow and Mullins, 2008), but it is not
known if defective buc mRNA localization reflects a direct role of
Buc protein in localizing its transcript or an indirect effect due to
absence of the Bb and oocyte polarity.
RNA-binding proteins (RNAbps), which can localize RNAs and
regulate their spatial and temporal translation or stability, are
attractive candidate regulators of buc localization and/or Buc protein
Development
Amanda E. Heim1, Odelya Hartung1, Sophie Rothhämel1,*, Elodie Ferreira1, Andreas Jenny1,2 and
Florence L. Marlow1,3,‡
activity. Indeed, several RNAbps, or their RNAs, localize to the Bbs
of zebrafish and frogs (Draper et al., 2007; Kloc et al., 2000; Kosaka
et al., 2007; Kroll et al., 2002; Marlow and Mullins, 2008; Song et
al., 2007; Zhao et al., 2001). Deleted in azoospermia-like (Dazl) is
a conserved RNAbp required for germ cell differentiation and
survival (Hashimoto et al., 2004; Houston and King, 2000; Houston
et al., 1998; McNeilly et al., 2000; Ruggiu et al., 1997; Saunders et
al., 2003). In Xenopus and zebrafish, dazl transcripts localize to the
Bb and later remain at the vegetal pole (Bontems et al., 2009; Chang
et al., 2004; Kloc et al., 2001; Kosaka et al., 2007; Maegawa et al.,
1999; Marlow and Mullins, 2008).
Like Dazl, RNA binding protein with multiple splice isoforms 2
(Rbpms2; also known as Hermes) is a conserved Bb-localized
RNAbp (Kosaka et al., 2007; Song et al., 2007; Zearfoss et al.,
2004). Rbpms2 colocalizes with and binds germ plasm RNAs
(Kosaka et al., 2007; Song et al., 2007) and has been postulated to
maintain their translational repression. Rbpms2 and dazl are not
localized in zebrafish buc mutants (Bontems et al., 2009; Marlow
and Mullins, 2008; Nojima et al., 2010). However, it has not been
determined whether Buc specifies the site of Bb assembly or
participates in recruiting proteins and RNAs to the Bb via indirect
or direct interaction.
Here we show that endogenous Buc protein is asymmetrically
localized in oocytes at stages before formation of the Bb, where Buc
later localizes; thus, Buc is the earliest marker of oocyte polarity in
zebrafish. Using a transgenic approach, we found that the buc
introns are required for full rescue of the egg polarity and axis
defects of bucp106/p106 mutant females. As with other localized
mRNAs, the buc 3′UTR harbors predicted recognition sites for
RNAbps. Transgenes encoding the full-length protein and 3′UTR
without introns (cbuc) cause ectopic Bb formation, whereas intronlacking versions of buc with a truncated 3′UTR (cbuc80) disrupt
animal-vegetal polarity. We show that Rbpms2 binds to buc but not
other Bb-localized RNAs, such as dazl. By contrast, Dazl binds buc
and dazl RNAs. Because Buc appears asymmetrically localized
prior to localization of its RNA, we postulate that a mechanism
involving localized translation or stabilization of Buc generates
asymmetry and allows recruitment of mRNAs via interactions
between Buc and RNAbps, such as Rbpms2. Our results indicate
that establishing oocyte polarity in zebrafish relies on precise
regulation of Buc levels and activity, possibly by a mechanism that
requires buc introns. Our findings suggest that Buc initiates a
positive-feedback mechanism whereby local production and/or
stabilization of Buc protein allows recruitment of more buc RNA
and, in turn, production of more Buc protein.
RESULTS
Asymmetric localization of Buc protein prior to Bb formation
To determine when and where endogenous Buc protein first
appears during oocyte development, we generated anti-Buc
antibodies. Endogenous Buc protein localized to the Bb in wildtype (WT) primary oocytes (Fig. 1A,A′,C-F; data not shown); no
localized Buc protein was detected in bucp106/p106 mutant oocytes
(Fig. 1B,B′) or when primary antibody was omitted (not shown).
Bb localization is consistent with the essential function of Buc in
forming this asymmetric oocyte structure. To investigate whether
asymmetric Buc protein might precede Bb formation and provide
an early marker of oocyte asymmetry, we examined earlier stages
of oogenesis. We detected asymmetrically enriched perinuclear
Buc protein at pre-Bb stages (Fig. 1G,G′), indicating that Buc
protein and zebrafish oocytes are polarized before Bbs are
detectable.
Development (2014) doi:10.1242/dev.090449
Fig. 1. Buc protein localizes to Balbiani bodies (Bbs) and is asymmetric
before Bb formation. (A,A′,C) Buc protein localization in Bbs (red arrows in
A,C) of stage Iaz (zygotene), Iap (pachytene) and Ib (larger than Ia, arrested
in diplotene) WT oocytes in whole-mount ovaries stained with anti-Buc
antibodies. Buc protein is not detected in bucp106/p106 mutants (B,B′). (A′,B′)
Tracings of the oocytes (blue lines) and their nuclei (yellow dashed lines)
from A and B. (D) DAPI-labeled nuclei of oocytes (yellow dashed circles) and
follicle cells (blue arrows). (E) Merge of C and D. (F) Corresponding phase
image to E. (G) Perinuclear localization of Buc (pink arrows) in WT stage Iap
oocytes before Bb formation. (G′) Tracing of oocytes (blue lines) and nuclei
(yellow lines). n/nuc, nucleus.
Intron-containing buc transgenes rescue egg polarity
phenotypes of buc mutants
Zebrafish maternal-effect mutants revealed that Buc protein is
essential for Bb formation, localization of buc and other Bb
mRNAs, and animal-vegetal (AnVg) axis formation (Bontems et al.,
2009; Dosch et al., 2004; Marlow and Mullins, 2008; Nojima et al.,
2010). To analyze the regulation and function of buc, we identified
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Development
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.090449
Fig. 2. buc transgenes with introns rescue buc egg
polarity phenotypes. (A,B) buc gene structure and the
constructs used herein. (A) The buc promoter was used to
express full-length buc or buc with a truncated 3′UTR.
(B) The full-length buc ORF containing the full 3′UTR
(cbuc) or a truncated buc 3′UTR (cbuc80) without introns:
grey, non-coding/intron; green, exon; light blue, 3′UTR. In
addition, a mutant Buc protein with the bucp106 nonsense
mutation was generated (cbucp10680). (C) Schematic and
genotyping assay. Products from genomic DNA are 90 bp
smaller in transgenes lacking introns. Gel images of
products from adult F1 progeny of cbuc80 founders.
(D,E) 8- to 16-cell stage F2 progeny of a gbuc rescued
mutant in different focal planes. Arrows in E indicate the
excess micropyles on embryos with rescued egg polarity.
(F) Clutch of gbuc rescued mutant female at 1 dpf.
(G) Higher magnification of embryos from F.
(H) Ventralized phenotypes of gbuc+ mutant females.
(I) Rescued progeny of a gbuc80 buc mutant founder.
(F-I) Rostral is left and caudal is right. (D-H) Progeny of F1
transgenic mothers. An, animal; Vg, vegetal.
buc promoter sequences that recapitulate buc expression
(supplementary material Fig. S1A,B) and determined that transgenic
reporters under control of the buc promoter are expressed in early
oocytes (supplementary material Fig. S1C-E).
We cloned a minigene including all introns (gbuc) and verified
that RNA from the transgene was properly spliced by RT-PCR,
sequence analysis and expression assays at stages when endogenous
buc transcripts were not detectable (supplementary material Fig. S2).
To facilitate rescue and structure-function analysis in buc mutants,
we generated transgenic lines expressing gbuc and various mutant
derivatives in buc heterozygotes (Fig. 2A-C). Only the gbuc
transgene rescued AnVg egg polarity and micropylar numbers in
progeny of bucp106/p106 mutants (Table 1, Fig. 2D-I). Some embryos
with rescued egg polarity had multiple micropyles (two to four on
one side), indicating incomplete rescue (Fig. 2E). Sixty-three percent
of rescued progeny were viable at 1 dpf and 92% of those showed
Genotype
No AnVg polarity,
multiple micropyles
WT polarity,
one micropyle
WT polarity,
multiple micropyles
Total
Tg[pBH:bucpromoter:gbuc]+; bucp106/+ F0-17
Tg[pBH:bucpromoter:gbuc]+; bucp106/+ F0-25
Tg[pBH:bucpromoter:gbuc]+; bucp106/+ F1-3
Tg[pBH:bucpromoter:gbuc]+; bucp106/+ F1-4
Tg[pBH:bucpromoter:gbuc]+; bucp106/+ F1-5
Tg[pBH:bucpromoter:gbuc]+; bucp106/p106 F1-1
Tg[pBH:bucpromoter:gbuc]+; bucp106/p106 F1-2
Tg[pBH:bucpromoter:gbuc]+; bucp106/p106 F1-6
Tg[pBH:bucpromoter:gbuc80]+; bucp106/p106 F0
Tg[bucpromoter:cbuc80]+; bucp106/+ F0
Tg[bucpromoter:cbuc80]+; bucp106/+ F0
Tg[bucpromoter:cbuc80]+; bucp106/+ F0
597 (30.2)
310 (35)
12 (8.5)
0 (0)
0 (0)
2 (1.5)
6 (6)
0 (0)
448 (77)
67 (15)
4 (15)
329 (39)
1371 (69.4)
583 (65)
105 (73.9)
60 (100)
27 (100)
129 (96.3)
72 (68)
43 (100)
99 (17)
389 (85)
23 (85)
523 (61)
8 (0.4)
0 (0)
25 (17.6)
0 (0)
0 (0)
3 (2.2)
28 (26)
0 (0)
34 (6)
0 (0)
0 (0)
0 (0)
1976
893
142
60
27
134
106
43
581
456
27
852
The percentage is shown in parentheses.
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Development
Table 1. Phenotypes of eggs of buc transgenic females
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Development (2014) doi:10.1242/dev.090449
normal morphology (Fig. 2F-H). As anticipated for a maternal-effect
gene, only half of these rescued progeny expressed the zygotic
bleeding heart reporter at 2 dpf, indicating that maternally supplied
gbuc rescued egg polarity in zygotes that did not inherit the
transgene (supplementary material Fig. S3). gbuc constructs with
truncated 3′UTRs (gbuc80) also rescued AnVg polarity, indicating
that the remaining 3′UTR sequence was sufficient to rescue egg
polarity (Table 1); however, only 18% of gbuc80 rescued embryos
were viable at 1 dpf and, of those, 36% were ventralized, indicating
rescue was incomplete and that 3′UTR sequences may contribute to
patterning (Fig. 2I; supplementary material Fig. S3).
buc transgenes disrupt egg polarity
Heterozygosity for the bucp106 allele alone does not disrupt egg
polarity (n>1900 eggs; 44 females) (Bontems et al., 2009; Marlow
and Mullins, 2008). Females that were WT or heterozygous bucp106/+
and positive for gbuc or transgenes lacking introns (cbuc and
cbuc80) produced two classes of progeny (Table 1, Fig. 3). One
class exhibited normal AnVg polarity, as indicated by cytoplasm at
the animal pole (Fig. 3A,C) and a single micropyle on their
eggshells as observed in WT (Fig. 3D). The second class resembled
buc mutants (Fig. 3B,C,E,I-K). Specifically, cytoplasm was detected
around the circumference of these eggs (Fig. 3B,L,M) and their
eggshells had excess numbers of micropyles (Fig. 3E). Chorion
elevation and egg size were comparable to WT, indicating that these
aspects of egg activation were normal (Fig. 3E,I-K). Polyspermy
was evident in eggs with excess micropyles based on DAPI staining
(data not shown). These data indicate that proper levels of Buc are
required for normal egg polarity.
Disrupting oocyte polarity causes excess micropylar cells
We conducted histological examination of the cbuc transgenic
ovaries to assess oocyte polarity. Morphologically, females
expressing transgenes lacking introns were normal and their ovaries
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Development
Fig. 3. buc transgenes without introns disrupt egg
polarity and follicle cell fates. (A-K) Dissecting microscope
images. (A-E) The sibling in A shows normal egg polarity at
high stage, whereas cytoplasm (black arrows) around the
circumference of siblings in B indicates lack of egg polarity.
(C) cbuc80 transgenic progeny resemble WT (yellow
asterisks) or lack polarity (blue asterisks); high stage.
(D) WT eggs (yellow arrow) have a single micropyle,
whereas (E) there are multiple micropyles on the eggshells
of progeny lacking egg polarity (n=1439 eggs), but not on
eggs with polarity (n=2349). Homozygous WT (F) or buc/+
heterozygous (G) females lacking the cbuc80 transgene
produce progeny with normal AnVg polarity, whereas (I,J)
sibling WT and buc/+ females with cbuc80 produce progeny
without AnVg polarity (blue asterisks). (H) buc mutants lack
AnVg polarity (K) even when cbuc80 is present.
(L) Quantification of phenotypes according to genotype and
transgene status. Each bar represents individual F1 or F2
females. The numbers correspond to the gel in
supplementary material Fig. S4. X, not in gel.
(M) Quantification of egg phenotypes of cbuc progeny of F1
females.
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.090449
Fig. 4. Defective Bb formation and excess
polarized somatic fates in transgenics lacking
introns. (A-D) Oocytes from cbuc80 transgenic
founders. (A-F) Hematoxylin and Eosin (H&E)stained F0 ovary sections reveal a normal
composition of oocytes, including (A) stage III
oocytes with single micropylar cells (arrow and
a1), (B) stage III oocytes with multiple micropylar
cells (arrows and b1 and b2), (C) stage I oocytes
with Bbs and (D) stage I oocytes lacking Bbs.
(E,F) Ectopic Bbs of cbuc+ F1 females. (GJ) DiOC6 staining of sectioned ovaries. cbuc80 F1
ovaries reveal primary oocytes with (G) and
without (H) Bbs. (I,J) Ectopic Bbs of cbuc+ F1
females. (K) BF view of oocyte in J. Arrows
indicate Bbs. (L) Quantification of Bbs from
different individual F1 transgenic females labeled
with DiOC6. Cg, cortical granules; n, nucleus.
Dominant phenotypes of buc transgenes
To further investigate whether cbuc transgenes disrupt Bb
development like buc mutants, we examined mitochondria and
846
endoplasmic reticulum (ER) in oocytes of cbuc transgenic females.
We observed three categories of stage Ib oocytes in WT females
expressing cbuc: normal, supernumerary Bbs, and those lacking Bbs
(Fig. 4E,F). Moreover, mitochondria and ER were detected in the
Bbs and excess Bbs of stage Ib oocytes (Fig. 4G-L) or were broadly
distributed (Fig. 4H; supplementary material Fig. S6), as in buc
mutants. By contrast, no ectopic Bbs were detected in cbuc80
females (Fig. 4C,D). Together, these data indicate that excess Buc
disrupts polarity and, in a 3′UTR-dependent manner, causes
supernumerary Bbs.
buc transgenes disrupt the localization of buc and other Bb
mRNAs
To determine whether transgenes lacking introns prevented
expression of endogenous buc, we used a qRT-PCR approach to
distinguish endogenous buc from transgenic transcripts. As
anticipated, both endogenous and cbuc80 transcripts were present in
transgenic ovaries and their progeny and transgene expression
correlated with the penetrance of egg polarity phenotypes (Fig. 5A).
In the most strongly affected cbuc ovaries, both transcripts were
reduced (Fig. 5A).
The cbuc80 construct contained only the first 80 bp of the 3′UTR
(Fig. 2B). This truncated 3′UTR retained predicted miR-302/371373/miR430 sites and lacked putative regulatory or protective
RNAbp sites (Fig. 2B). To determine if cbuc transgenes affect RNA
Development
were composed of oocytes of all stages. As predicted based on their
eggs, cortical granule distribution and yolk accumulation in
advanced stage oocytes were normal (Fig. 4A,B). However,
consistent with the excess micropyles of eggs with defective
polarity, we observed two populations of advanced stage oocytes,
those with one somatic micropylar cell positioned at the animal pole
(Fig. 4Aa) and those with multiple micropylar cells (Fig. 4Bb1,b2).
The micropyle and egg polarity phenotypes were coincident in
females expressing cbuc and cbuc80. No Buc protein expression
was detected in the follicle cell layer of bucp106/+ females negative
or positive for cbuc transgenes (supplementary material Fig. S5).
Furthermore, the Tg[buc:mApple] promoter reporter showed
mApple fluorescence only in oocytes (supplementary material Fig.
S1C-E′′′). These results indicate that buc in the germline can nonautonomously influence the otherwise ‘wild-type’ somatic cells,
either by changing their fate or permitting survival of micropyle
progenitors. Because the two classes of oocytes are intermixed
within the same ovary in cbuc and cbuc80 transgenics, the oocyte
signals that regulate micropyle numbers apparently act at short
range, such that one oocyte does not affect the mycropylar cells
associated with neighboring oocytes.
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Development (2014) doi:10.1242/dev.090449
localization we examined buc transcript localization by wholemount in situ hybridization using a probe that detects both
endogenous and transgenic buc transcripts. Consistent with the RTPCR analysis, buc transcripts were present in transgenic ovaries. As
anticipated based on the lack of a detectable Bb, buc, vasa and other
RNAs were not asymmetrically localized in cbuc80 oocytes lacking
polarity (Fig. 5B-G; data not shown), a phenotype reminiscent of
buc mutants (Bontems et al., 2009; Marlow and Mullins, 2008). We
performed qRT-PCR on sorted oocytes and determined that cbuc80
and endogenous buc transcripts were present throughout Bb
dispersal (Fig. 5H,I). These data indicate that endogenous buc
mRNA was not maintained at late stages as a consequence of failure
to properly localize buc transcripts, as occurs in buc mutants.
Functional Buc protein is required for dominant phenotypes
If Buc protein acts primarily to seed the assembly of a polarity
complex, we reasoned that excess or mislocalized Buc protein might
produce polarity phenotypes similar to buc mutants (Bontems et al.,
2009; Marlow and Mullins, 2008). To determine if cbuc phenotypes
were due to excess or mislocalized Buc protein, we examined Buc
distribution in oocytes of various buc genotypes with and without
cbuc or cbuc80. Whereas Buc protein localized to the Bb of
heterozygotes lacking cbuc or cbuc80 (Fig. 6A), it was not
asymmetric in heterozygotes expressing cbuc80 (Fig. 6B).
Homozygous WT and heterozygous females (Fig. 6C) showed Buc
protein within the Bb of nearly all primary oocytes examined. By
contrast, stage Ib oocytes in WT or heterozygous bucp106/+ females
expressing cbuc80 lacked asymmetric Buc protein and resembled
bucp106/p106 mutant oocytes, which lacked polarity whether positive
for cbuc80 or not (Fig. 6C).
To functionally assess whether ectopic Buc protein or its mRNA
disrupted polarity, we analyzed females with an analogous transgene
containing the bucp106 nonsense mutant allele (cbucp10680)
(Fig. 2B, Table 2). Significantly, this nonsense mutation does not
cause phenotypes in bucp106/+ heterozygotes (Bontems et al., 2009;
Marlow and Mullins, 2008). In contrast to females expressing
cbuc80, cbucp10680 F0 and F1 transgenic females produced only
progeny with normal egg polarity and single micropyles (Table 2).
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Development
Fig. 5. buc transgenes lacking
introns disrupt RNA
localization. (A) RT-PCR on
ovary lysate cDNA. Transgenic
and endogenous transcript
expression in ovaries of F1
transgenic females expressing
intron-lacking transgenes in
bucp106/+ heterozygotes.
(B,C) buc RNA in ovaries of
cbuc80+;bucp106/+ F1 transgenic
females (B) resembles WT or (C)
is not asymmetrically localized.
(D) Quantification of buc RNA
expression patterns in stage I
oocytes. (E,F) vasa expression in
(E) F1 transgenic– and (F)
cbuc80 F1 transgenic+ ovaries.
(B,E) Blue arrows indicate the
Bb. (G) Quantification of vasa
expression patterns. (HJ) Relative expression of (H)
cbuc80, (I) endogenous buc and
(J) vasa in sorted oocytes of F1
or F2 females. (D,G) het,
bucp106/+; mut, bucp106/p106; wt,
buc+/+.
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Development (2014) doi:10.1242/dev.090449
Founder females
FO bucp106/+;Tg[buc:cbucp10680]+
FO bucp106/+;Tg[buc:cbucp10680]+
FO bucp106/+;Tg[buc:cbucp10680]+
FO bucp106/+;Tg[buc:cbucp10680]+
F1 adult females
F1 buc+I+;Tg[buc:cbucp10680]+
F1 buc+I+;Tg[buc:cbucp10680]+
F1 bucp106/+;Tg[buc:cbucp10680]+
F1 bucp106/+;Tg[buc:cbucp10680]+
F1 bucp106/+;Tg[buc:cbucp10680]+
Fig. 6. Antimorphic activity of buc transgenes lacking introns requires
functional protein and disrupts endogenous Buc. (A,B) Sectioned and
stained oocytes. Buc protein localizes to the Bb (blue arrow) of (A)
bucp106/+;cbuc80– stage Ib oocytes, and is not asymmetric in (B)
bucp106/+;cbuc80+ stage Ib oocytes. (C) Quantification of Bbs of Buc-labeled
oocytes of F1 or F2 females. n, the number of oocytes examined.
As for cbuc80, we analyzed cbucp10680 transcripts and found that
its RNA was expressed at a comparable level (Fig. 5A). By contrast,
bucp106/+ heterozygous transgenic females generated from crosses of
two cbucp10680 transgenic F1s produced progeny lacking AnVg
polarity (26%; n=1183 eggs, 13 females), whereas their buc+/+
sibling transgenic females infrequently produced progeny lacking
egg polarity (2%; n=1021 eggs, 13 females). This indicates that
higher copy numbers of cbucp10680 can disrupt AnVg polarity.
Taken together, these data indicate that the dominant polarity
phenotypes of cbuc80 were due to a functional coding sequence for
Buc protein in the transgene.
Buc protein interacts with conserved germ plasmassociated RNAbps
Previous studies have shown that buc mRNA localizes to the Bb and
an exogenous GFP-Buc fusion expressed from RNA injected into
oocytes accumulates in the Bb (Bontems et al., 2009). Here we show
that endogenous Buc protein localizes to the Bb (Fig. 1) via a
mechanism that is likely to involve local production or stabilization
of Buc protein, because endogenous buc transcripts are not
asymmetric before Bb formation or in buc mutants (Bontems et al.,
2009). The lack of Bbs in buc mutants demonstrates that Buc is
required to assemble this conserved aggregate of RNAs and
proteins, but the mechanism of Buc action is not known. One
potential mechanism is that Buc mediates Bb assembly and RNA
localization by interacting with RNAbps. Rbpms2 is a conserved
RNAbp that contains two RNA recognition motif (RRM) domains
(Gerber et al., 1999; Kosaka et al., 2007; Song et al., 2007; Zearfoss
et al., 2004). Rbpms2 protein localizes to the Bbs of oocytes in
848
No AnVg polarity
WT polarity Total
0
0
0
0
266
164
191
97
266
164
191
97
0
0
0
0
0
388
390
116
49
39
388
390
116
49
39
zebrafish (Kosaka et al., 2007; Marlow and Mullins, 2008) and frogs
(Zearfoss et al., 2004) and is not localized in buc mutants (Marlow
and Mullins, 2008). Based on their localization, we hypothesized
that Buc might bind to Rbpms2, which could then, via its RNAbinding motifs, recruit or retain other Bb RNAs (such as buc
mRNA). To test this possibility, we performed yeast two-hybrid
(Y2H) experiments. Indeed, full-length Buc (Buc-Fl) interacted with
zebrafish and human Rbpms2, but not with another RNAbp, DAZ
associated protein 1 (Dazap1) (Fig. 7A,B; data not shown) (Claussen
and Pieler, 2004; Kurihara et al., 2004).
To confirm the interaction observed in yeast, we transfected
HEK293 cells with GFP-Buc or YFP-Diego, an unrelated bait, and
Myc-Rbpms2 or Myc-Dishevelled as a control, and conducted coimmunoprecipitation (co-IP) experiments on the HEK293 cell
lysates using anti-GFP antibody. We found that Rbpms2 coimmunoprecipitated with GFP-Buc, but not YFP-Diego (Fig. 7C).
To independently assess binding, we conducted in vitro GST pulldown assays. We found that GST-Buc fusion protein interacted with
35
S-labeled Rbpms2 (Fig. 7D). These approaches validated the
interaction between Buc and Rbpms2 that we observed in the Y2H
assay and indicate that Buc is likely to directly bind to Rbpms2.
We used the binding interaction between Buc and Rbpms2 to
identify potential functional domains of the Buc protein. Using the
Y2H assay, we mapped the Rbpms2 binding site using six partially
overlapping Buc truncations covering the full protein (Fig. 7A,B;
data not shown). Two truncations were N-terminal Buc deletions
(BucΔ1-252 and BucΔ1-386), two carried nonsense mutations
corresponding to the bucp43 and bucp106 mutant alleles (Bontems et
al., 2009), and two harbored internal deletions [BucΔ53-116 frame
shift (fs) and BucΔ353-599]. BucΔ1-252 and BucΔ1-386 did not
interact with Rbpms2, but the Bucp43 and Bucp106 mutant proteins
did. The internal deletions displayed an intermediate binding
phenotype in the Y2H assays. Together, these data indicate that the
Buc N-terminus mediates binding to Rbpms2 and that the internal
region of the protein either augments or stabilizes this interaction.
Rbpms2 has been previously reported to interact with germ plasm
components in Xenopus (Song et al., 2007). Colocalization of Buc
and Rbpms2 in the Bb along with germ plasm RNAs and the
interaction between Buc and Rbpms2 suggest a mechanism whereby
Buc interaction with Rbpms2 could recruit buc and other Bb RNAs.
Two Bb-localized RNAbps, Dazl and Rbpms2, bind buc RNA
Colocalization of Buc and Rbpms2 in the Bb along with germ plasm
RNAs and the direct interaction between Buc and Rbpms2 suggest a
mechanism whereby Buc interaction with Rbpms2 recruits RNAs to
the Bb. To explore this hypothesis, we investigated whether Rbpms2
Development
Table 2. Egg phenotype analysis indicates that cbucp106/+ F0 and
F1 females produce normal eggs
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.090449
and another Bb RNAbp that associates with germline RNAs in
primordial germ cells, Dazl (Kosaka et al., 2007; Takeda et al., 2009),
bind to Bb RNAs in oocyte lysates using Myc-tagged Rbpms2 (MTRbpms2) and Myc-tagged Dazl (MT-Dazl) as baits (Fig. 7E). Among
the ovary transcripts examined in the IP between MT-Rbpms2, we
only detected buc (Fig. 7E). buc was also detected from IP with MTDazl. In addition to detecting buc in association with MT-Dazl, we
also detected dazl, which did not bind Rbpms2 or beads alone
(Fig. 7E). These results indicate that Rbpms2 and Dazl bind to some
Bb mRNAs and might mediate their recruitment to the Bb.
DISCUSSION
The vertebrate AnVg axis is established during oogenesis. Oocyte
polarity is a prerequisite for determining the prospective embryonic
849
Development
Fig. 7. Buc protein interacts with the RNAbp Rbpms2.
(A) Summary of buc deletion constructs used in the yeast twohybrid analyses in B. Buc interacts with Rbpms2 via the Nterminus of Buc. The transformation (TF) control plates select
for bait (pDBD) and prey (pAD) plasmids, whereas the
interaction plates select for binding between the bait and prey
proteins. Control baits and preys were KrevI and a strongly
interacting prey Ral/GDS (wt) and two mutants: Ral/GDSm1
(moderate/medium interaction with Krev) and Ral/GDSm2
(weak/no interaction with KrevI). (C) Rbpms2 coimmunoprecipitates with Buc in HEK293 lysates. Top panels
indicate transfected plasmids. Long exposure reveals proteins
with lower expression levels. (D) Pull-down assay with GSTand 35S-labeled GFP fusion proteins. (pd, pull down). GST
fusion protein inputs were visualized with Coomassie Blue.
(E) RNA IP experiments using in vitro synthesized Myc-tagged
RNAbps. The RNAs that co-immunoprecipitated with Myctagged RNAbps were amplified by RT-PCR. MT-Rbpms2
immunoprecipitated buc but not nanos2, vasa or dazl. MT-Dazl
associated with buc and dazl mRNAs.
axes and setting aside the germ cell determinants in some nonmammalian vertebrates (Abrams and Mullins, 2009; Marlow, 2010;
Mir and Heasman, 2008; Schier and Talbot, 2005). The Bb is an
evolutionarily conserved asymmetric structure present in early
oocytes of all animals examined, including humans. Our study
identifies Buc protein, an essential regulator of the AnVg axis, as
the earliest marker of oocyte polarity in zebrafish. Discrete
localization of endogenous Buc protein and overexpression
phenotypes caused by transgenes with intact open reading frames
indicate that spatial or temporal regulation of Buc translation or
stabilization is essential for oocyte polarity. Interestingly, we also
find a non-autonomous role of the germline in limiting somatic
follicle cell fates, pointing toward communication between the
germline and somatic cells. Further, we identify interactions with the
RNAbps Rbpms2 and Dazl and buc products. Buc protein and buc
RNA bind Rbpms2, whereas buc and dazl RNAs bind to another Bb
component, Dazl. Thus, interactions with RNAbps might play key
roles in regulating Buc activity and establishing oocyte polarity in
zebrafish.
Regulation of buc RNA is likely to generate asymmetric Buc
protein localization and trigger Bb assembly
Endogenous Buc protein is localized asymmetrically before Bb
assembly and later is localized to the Bb. At the stages when
asymmetric perinuclear Buc protein is detected, its transcripts
remain broadly distributed, indicating that local stabilization of Buc
or selective translation of its mRNA, rather than global
redistribution of buc RNA, is likely to initiate asymmetric Buc.
Mechanisms to generate asymmetric protein localization include
localized translation, local stabilization and aggregation, and active
transport of a protein to a specific subcellular location (Gagnon and
Mowry, 2011; Hachet and Ephrussi, 2001; Holt and Bullock, 2009;
Kloc and Etkin, 2005; Kugler and Lasko, 2009; Minakhina and
Steward, 2005; St Johnston, 2005; Zhou and King, 2004). If Buc
protein were uniformly produced and then transported to a
perinuclear position, we would expect to initially detect Buc protein
throughout the oocyte followed by progressive enrichment adjacent
to the nucleus. Our data argue against such a model, although we
cannot exclude that Buc is present throughout the cell at levels
below our detection. Moreover, asymmetric localization of Buc
protein in WT and the loss of oocyte polarity caused by the buc
transgenes seem more consistent with a mechanism that involves
localized translation or stabilization of Buc protein. According to the
localized stabilization scenario, broadly produced Buc protein would
be rapidly degraded except near the nucleus where it accumulates.
Alternatively, or in addition, the initially ubiquitous endogenous buc
mRNA could be translationally repressed via association with
RNAbps except near the nucleus, where a limiting or localized
factor(s) would alleviate repression of buc RNA. Such a mechanism
would explain the gain-of-function phenotypes that we observe in
WT (buc+/+; bucp106/+) genotypes expressing gbuc and cbuc
transgenics. This would also be consistent with the potentially
dominant-negative phenotypes caused by high doses of cbucp10680,
which might result from titration of a limiting repressor and
premature translation of endogenous buc analogous to the
mechanisms reported for oskar mRNAs harboring stop codons
(Zimyanin et al., 2007). The identity of the protein(s) regulating buc
translation remains to be determined. Vasa is a compelling
candidate, as it is known to promote translation in the female
germline of flies (Carrera et al., 2000; Lasko and Ashburner, 1988;
Markussen et al., 1995; Styhler et al., 1998) and Vasa protein is
perinuclear in early stage zebrafish oocytes (Knaut et al., 2000). In
850
Development (2014) doi:10.1242/dev.090449
WT oocytes, however, Vasa protein is not localized to the Bb in
zebrafish (Knaut et al., 2000), suggesting that other, yet-to-beidentified proteins would regulate Buc translation within the Bb.
Oocyte polarity and follicle cell fate
In WT oocytes there is a single animal pole where only one
micropyle develops. In buc mutants, expanded or ectopic animal
poles, as evidenced by multiple domains of the animal pole marker
vg1 (igf2bp3 – ZFIN) (Marlow and Mullins, 2008), support
development of excess micropylar cells, indicating that the animal
pole environment might provide an instructive or permissive cue for
micropylar cell survival or fate. Notably, we observed eggs with WT
polarity and two to four micropyles on one half of the eggshell of
some gbuc rescued mutant eggs. In cbuc80 and cbuc transgenic eggs
without polarity, we always observed supernumerary micropyles.
Similarly, all eggs from cbuc80 and cbuc transgenic females with
normal polarity had only one micropyle. Concordance between egg
polarity and micropyle phenotypes and the observation that follicle
cells express no detectable Buc protein are consistent with a model
whereby local oocyte signals regulate micropyle cell fate. The nature
of the communication is not clear, but it is likely to involve a closerange signal, possibly direct cell contact, rather than a broadly
diffusible signal, because oocytes with multiple micropyles can be
adjacent to those with one micropylar cell.
A self-organizing mechanism to recruit RNAs to the Bb
Buc protein is essential for RNA localization along the zebrafish
AnVg oocyte axis, including for the earliest RNAs, which localize
to the Bb near the prospective vegetal pole (Bontems et al., 2009;
Marlow and Mullins, 2008; Nojima et al., 2010). Buc harbors no
known RNA-binding motifs, but our analysis supports the
possibility that Buc exerts its effects on RNA by serving as a
scaffold and assembly factor for RNAbps. We identified a novel
interaction between Buc protein and a conserved Bb-localized
RNAbp Rbpms2 (Kosaka et al., 2007; Song et al., 2007).
Interaction between Buc and Rbpms2 requires the N-terminus of
Buc protein. Thus, our work defines a potential functional domain
of Buc protein and, intriguingly, a potential mechanism by which
Buc could recruit RNAs to the Bb. Our data suggest that Buc
might promote oocyte polarity by interacting with RNAbps to
direct the assembly of ribonucleoprotein (RNP) complexes to form
the Bb (Fig. 8).
Many Bb-localized RNAs contain a mitochondrial cloud
localization element (MCLE) site, which is sufficient to direct RNAs
to the Bb in Xenopus oocytes (Kosaka et al., 2007). The Xenopus
MCLE includes six copies of a hexamer sequence, but zebrafish buc
RNA apparently has only one copy, as is also the case for zebrafish
nanos3 and dazl. Although the single hexamer sequence is present
in cbuc80, this region is not sufficient for proper regulation and
localization of buc. Although several RNAs, predominantly germ
plasm RNAs, localize to the Bb of early oocytes, these RNAs
occupy distinct cellular compartments in late stage oocytes. For
example, transcripts of nanos, vasa, buc, dazl and dorsal axis
regulators including syntabulin and wnt8a all localize to the Bb of
stage I oocytes (Bontems et al., 2009; Draper et al., 2007; Knaut et
al., 2000; Kosaka et al., 2007; Lu et al., 2011; Nojima et al., 2010),
but by stage III buc transcripts are localized to the animal pole,
nanos is distributed throughout the oocyte, vasa is circumferential
at the cortex, and dazl and syntabulin remain at the vegetal pole. For
many germ plasm RNAs examined in zebrafish oocytes, the 3′UTR
is sufficient to achieve their proper localization (Kosaka et al.,
2007).
Development
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.090449
Fig. 8. A feedback amplification model for Bb assembly. (A) Model depicting how Buc might promote oocyte polarity and Bb formation via interactions with
RNAbps. (B) Potential events in cbuc transgenics. We hypothesize that endogenous buc transcripts are loaded with a repressor prior to nuclear export. Since
cbuc RNA is not spliced, the repressor is not loaded and cbuc transcripts are translated ectopically or prematurely. Alternatively and independently of splicing,
a limiting repressor might be overwhelmed by excess buc transcripts in transgenic oocytes. Either way, ectopic foci of Buc protein would recruit Bb components
and produce the multiple small Bbs observed with DiOC6 and H&E. In cbuc80 transgenics, we hypothesize that either cbuc80 transcripts are not recruited or
might not be translated as efficiently due to their lack of a 3′UTR. Consequently, local concentrations of Buc protein may not be sufficient to support the
development of ectopic Bbs. Based on their smaller size, their perinuclear proximity and the eventual egg polarity defects, we hypothesize that the ectopic Bbs
of cbuc transgenic oocytes do not reach the cortex, resulting in failure to specify the vegetal pole and a lack of animal-vegetal polarity.
2007). Transport of RNA to the vegetal pole via the Bb has been
proposed to involve an entrapment and expansion mechanism
(Chang et al., 2004; Wilk et al., 2005). A self-organizing and
amplifying Buc/Balbiani complex could facilitate robust recruitment
(entrapment) of RNP complexes, including those containing buc.
Localized translation of buc could drive further recruitment of RNPs
and expansion of the Bb. Buc might associate with a discrete subset
of RNAbps that are capable of forming multiple distinct RNPs.
Alternatively, Buc could recruit all Bb-localized RNAs/RNPs, which
could then be sorted within the Bb. Either mechanism would be
sufficient to recruit the diversity of RNPs that are anticipated to
comprise the Bb based on the unique mechanisms (3′UTR versus
splicing mediated) that generate distinct and dynamic localization
patterns of RNAs, including buc, that transit through the Bb in
zebrafish.
A Buc-mediated feedback amplification mechanism to
establish oocyte polarity
Localized Buc protein and its association with RNAbps provides a
mechanism to recruit Bb RNAs, including buc. Based on the
localization of Buc protein and the dominant phenotypes of the cbuc
transgenes, we hypothesize that an RNAbp could specifically interact
with the spliced buc mRNA to prevent ectopic translation of buc
851
Development
Similar to the germ plasm RNAs that have been examined,
patterning molecules, including wnt8a and syntabulin, localize to the
Bb. syntabulin is localized by a Buc-dependent pathway. The full
exon-intron structure of syntabulin is required for its proper
localization and activity (Nojima et al., 2010). Like syntabulin, buc
RNA localizes to the Bb (Bontems et al., 2009) and we show that
full rescue of the buc mutant phenotypes requires the buc introns.
Notably, incompletely rescued buc mutants are ventralized, possibly
due to incomplete rescue of dorsal determinant localization in these
oocytes. In Drosophila oocytes, the exon junction complex (EJC)
has been proposed to regulate Gurken (TGFα) signaling between
posterior follicle cells and the oocyte during axis formation, and to
mediate oskar mRNA localization and translation during germ cell
determination (Hachet and Ephrussi, 2001; Micklem et al., 1997;
Mohr et al., 2001; Newmark and Boswell, 1994). Our finding that
buc minigene constructs must have introns to rescue buc mutants,
together with previous studies of syntabulin (Nojima et al., 2010),
indicate that the EJC might be involved in Buc-mediated Bb
development and transport.
Although it is not clear how RNAs are directed or selected for Bb
localization, it is clear for those RNAs that have been examined that
early localization to the Bb is prerequisite for their proper
localization at later stages of oocyte development (Kosaka et al.,
RESEARCH ARTICLE
MATERIALS AND METHODS
Antibodies and immunostaining
YenZym (San Francisco, CA, USA) custom antibody service was used to
raise rabbit polyclonal antibodies against Buc epitopes: residues 1-15
MEGINNNSQPMGVGQ (Y1165 and Y1166) and residues 602-617
KSIHQQRPRSEYNDY (Y1163 and Y1164).
Ovaries were dissected from adults, fixed in 4% paraformaldehyde (PFA),
washed in PBS, dehydrated in methanol, rinsed and stained as described
(Marlow and Mullins, 2008). Primary Buc antibodies were diluted 1:500.
Secondary antibodies (anti-rabbit Alexa Fluor 488 or 546; Molecular
Probes) were diluted 1:500. Vectashield (Vector Labs) containing DAPI was
used to label the nuclei.
Mitochondria and ER were visualized by staining with DiOC6 [Molecular
Probes, D-273; 0.5 μg/ml in PBS+Tween (PBST):dimethyl sulfoxide] at
room temperature followed by PBST washes (Marlow and Mullins, 2008).
Images were acquired using a Zeiss Axio Observer inverted microscope
equipped with Apotome and a CCD camera.
Plasmid construction, transient assays and transgenesis
The ~2 kb buc promoter fragment was amplified from genomic DNA
(primers in supplementary material Table S1) and cloned into the Gateway
pCR8 entry vector (Invitrogen) and sequenced (Macrogen). Gateway
adapters were added using the buc 2Kprom attB4 and buc prom attB1R
primers (supplementary material Table S1) to generate the p5E-buc
promoter.
The buc open reading frame (ORF) was amplified from ovary cDNA
using the Invitrogen SuperScript III reverse transcriptase kit with oligo(dT)
primers and the primers listed in supplementary material Table S1 to obtain
the full-length (cbuc) and 3′UTR deletion (cbuc80) constructs. The
amplified buc ORF was cloned into pCR8 and sequenced (Macrogen).
Rescue plasmids were cloned into pCR8 and recombined into pCS2+
derived Gateway destination vectors (Kawakami, 2005; Kawakami, 2007;
Kwan et al., 2007; Villefranc et al., 2007) to generate
pTolbuc:bucORFfull3′UTR (cbuc), pTolbuc:bucORF80bp3′UTR (cbuc80),
pTolbuc:bucp106ORF80bp∆3′UTR (cbucp106) and buc-intron-exon (gbuc)
852
clones. Transgenic fish were generated by injecting 25-50 pg plasmid DNA
plus 25-50 pg transposase RNA into bucp106/+ heterozygotes.
pBH-R4/R2 was generated by modifying the ‘bleeding heart’ Tol2 plasmid
(pBH) from pBH-mcs(multi-cloning site) (a gift from Michael L. Nonet,
Washington University St Louis) to include a Gateway-compatible
attR4/attR2 cassette, namely attR4-chloramphenicol resistance-ccdB
survival gene-attR2 from pTolDestR4R2 (Villefranc et al., 2007).
The buc promoter reporter construct was generated in pBH-R4/R2. The
p5E-buc promoter was recombined with pME-mApple (Tol2 kit, v2.0) into
pBH-R4/R2 (Invitrogen). This plasmid was injected (50 pg) along with
transposase RNA (25 pg) to generate transgenic lines.
Genotyping
Genomic DNA was isolated from fin clips. Linked SSLP markers were used
to genotype for buc (Bontems et al., 2009; Knapik et al., 1998).
In situ hybridization and histology
Females were anesthetized in Tricaine as described (Westerfield, 1995) and
the ovaries were dissected. Whole-mount in situ hybridization was
performed as described previously (Thisse and Thisse, 1998) except that
hybridization was at 65°C and BM purple AP (Roche) was used. In situ
probes: the buc ORF was amplified with the primers listed in supplementary
material Table S1 and cloned into pCS2; vasa was described previously
(Yoon et al., 1997). Images were acquired using an AxioPlan2 or AxioSkop2
microscope equipped with an AxioCam CCD camera (Zeiss) or an Olympus
SZ16 fluorescent dissecting microscope and Microfire digital camera
(Olympus). Images were processed in ImageJ (NIH), Adobe Photoshop and
Adobe Illustrator.
For Hematoxylin and Eosin (H&E) staining, dissected ovaries were fixed
in 4% PFA, washed in PBS, dehydrated in methanol, then embedded in
paraffin and sectioned. Deparaffinized slides were stained in H&E, coated
with Permount solution (Fisher Scientific), coverslipped, and imaged using
an AxioSkop2 microscope and AxioCam CCD camera.
Oocytes were staged according to Selman et al. (Selman et al., 1993).
RT-PCR
Ovaries and other tissues (supplementary material Fig. S1A) were dissected
from the specified genotypes. Oocytes were sorted according to Selman et
al. (Selman et al., 1993). Trizol (Life Technologies)-extracted RNA was used
for oligo(dT) cDNA preparation (using Invitrogen SuperScript III reverse
transcriptase) and RT-PCR was performed using the primers listed in
supplementary material Table S1.
Yeast two-hybrid assays
The ProQuest System (Invitrogen) was used for Y2H assays. Baits and preys
were prepared from ovary cDNA as described above (for primers see
supplementary material Table S1), cloned into pCR8, sequenced, then
recombined into pDEST32 or pDEST22 vectors.
Immunoprecipitation and GST pull-downs
HEK293 cells (1×106) were transfected with 3 μg pCMV-DshMyc, pCSYFP-Dgo (Boutros et al., 1998; Jenny et al., 2005) or pCS2-MTRbpms2 or
pCS2-GFP-Buc overnight with 3:1 polyethylenimine:DNA. IP was with 1
μg of anti-Myc antibodies (9E10, Santa Cruz) (Jenny et al., 2005).
Precipitated proteins were separated by SDS-PAGE, transferred to
ImmobilonP (Millipore) and processed for ECL detection (GE Healthcare).
Short exposures comprised 1 minute and long exposures 10-15 minutes.
GST proteins were purified as described previously (Jenny et al., 2003).
1 μg DNA was translated using the coupled in vitro transcription-translation
system (Promega) with 35S. For GST pull-downs, GST fusion protein (5 μg)
was bound to 15 μl GST-Sepharose (Amersham), washed, incubated with
35
S-labeled proteins (5 μl) for 1 hour and analyzed as described (Jenny et
al., 2003).
RNA immunoprecipitation
Ovaries were dissected, snap frozen and stored (−80°C). According to Song
et al. (Song et al., 2007), ovaries were homogenized (1 ml YSS buffer) and
Development
transcripts. Alternatively, or in addition, a limiting RNAbp might
maintain repression of buc RNA. It is also possible that Buc
accumulates via localized stabilization. Either way, after Buc protein
accumulates asymmetrically adjacent to the nucleus, it can interact
with its binding partners, such as Rbpms2, which could then recruit
their cognate RNAs to form the Bb. Once Buc protein initiates Bb
assembly, RNAs are recruited to the Bb by interaction with RNAbps
that localize there, including Dazl and Rbpms2 (Kosaka et al., 2007;
Song et al., 2007). Some RNAs recruited to the Bb, like vasa, may be
silenced there [as Vasa protein, in contrast to its mRNA, is not a
component of the Bb (Knaut et al., 2000)]. However, others, including
buc and dazl, may be translationally activated within the Bb to sustain
its development (Bontems et al., 2009; Kosaka et al., 2007). In the
case of Buc, this feedback amplification would drive further localized
production of Buc protein and expansion of the Bb. The localization
and apparent abundance of Buc protein within the Bb, failure to
assemble the Bb and recruit buc RNA in buc mutants (Bontems et al.,
2009; Marlow and Mullins, 2008), and the protein- and 3′UTRdependent dominant phenotypes caused by cbuc and cbuc80
transgenes, are consistent with a Buc-dependent recruitment feedback
amplification mechanism acting to establish the initial asymmetry in
the oocyte (Fig. 8B,C). In this model, buc mRNA is initially present
throughout the oocyte, but Buc protein is only translated or stabilized
in a small region. Local accumulation of Buc protein establishes the
position of the Bb and recruits RNAbps that in turn recruit buc and
other RNAs to the Bb. Such a feedback mechanism that recruits buc
mRNA and promotes further accumulation of Buc protein and other
RNAs and proteins could establish AnVg polarity and lay the
foundation for the later forming embryonic axes.
Development (2014) doi:10.1242/dev.090449
centrifuged. The pellet and supernatant were retained. 250 μl of resuspended
pellet was pre-cleared with Myc beads (30 μl; Clontech, 631208) for 1 hour
at 4°C. Pre-cleared lysate was added to pCS2-MT-protein reticulocyte lysate
(45 μl) plus Myc beads (30 μl) and incubated (1 hour at 4°C). Beads were
washed (YSS buffer), then incubated in proteinase K lysis buffer (100 μl)
and proteinase K (10 μg) (1 hour at 50°C). RNA was isolated using Trizol
and precipitated (3 M sodium acetate pH 4.5 in ethanol). Precipitated RNA
was used for cDNA synthesis. cDNA (0.5 μg) was used for RT-PCR analysis
(primers listed in supplementary material Table S1).
Acknowledgements
We are grateful to Lilianna Solnica-Krezel, William S. Talbot and members of the
F.L.M. laboratory for discussions and manuscript evaluation. We thank S. Kalinin
and C. Depaolo for fish care, and the Einstein Histopathology Core for histology
services.
Competing interests
The authors declare no competing financial interests.
Author contributions
A.E.H., O.H. and F.L.M. performed transgenic construction, phenotypic and
molecular analyses. S.R. and A.J. performed Y2H/protein interactions. A.E.H. and
E.F. performed RNA IP. All authors discussed data and the manuscript. F.L.M.
wrote the manuscript.
Funding
This work was supported in part by National Institutes of Health grants
RO1GM089979 and start-up funds to F.L.M.; T32-GM007288 to O.H.; and
RO1GM088202 to A.J. Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.090449/-/DC1
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